During an average year, 10-20 percent of the US population becomes infected with influenza virus, but the only antiviral treatment available for those infected with influenza A virus is rimantadine, the target for which is the M2 ion channel protein. More effective inhibitors of the channel are needed because rimantadine-resistant mutants are generated rapidly. In the previous grant period we found that inhibition of the channel occurs when the pH of the cytoplasmic domain is lowered and transition metal complexes are formed with the M2 molecule. We propose to determine the mechanism for these inhibitions in order to identify new targets for drug action on the M2 molecule. From functional measurements we have found that the active state of the M2 ion channel is a homotetramer and we have identified residues that line the channel pore. We propose to determine the structure of the molecule with direct measurements because this structural information will allow for the design of more useful inhibitors. The individual aims are: 1. To understand the mechanism for inhibition of M2 ion channel as a result of decreased pH of the C-terminal domain. 2. To determine the M2 ion channel pore structure with the substituted cysteine accessibility method. 3. To use transition metal complexes to probe the pore structure of the M2 ion channel. 4. To apply the approach of site-directed spin labeling of the M2 ion channel transmembrane domain to determine its resting structure and structural changes that occur on activation by low pH. 5. To understand the role of transmembrane histidine 37 in proton transport by the M2 ion channel. 6. In a separate set of experiments we will test the hypothesis that the NB protein of influenza B virus functions as an ion channel.